Antibodies have been used to treat cancer and immunological or vascular disease. Antibody—medicament conjugates allow targeted delivery of the medicament moiety to the tumor and other diseased tissue, and systemic administration of unconjugated pharmaceutical agents may bring unbearable levels of toxicity to normal cells.
Basic unit of Natural antibodies is a monomer, which is composed of two identical heavy chains and two identical light chains connected by disulfide bond. There are at least five different types of heavy chains, i.e. γ, α, δ, μ and ε, which provide different effector functions. Heavy chains γ, α and δ have three constant domains (CH1, CH2 and CH3), heavy chains μ and ε have four constant domains (CH1, CH2, CH3 and CH4). Each heavy chain also has a variable domain (VH). There are at least two types of light chain, i.e., λ and κ, wherein each light chain comprises a constant domain (CL) and a variable domain (VL).
According to the amino acid sequence of the heavy chain constant domain, native human antibody can be classified into five categories: IgG IgA, IgM, IgD and IgE. Several categories of these categories can be further divided into subclasses or isotypes, such as IgG1, IgG2, IgG3, IgG4, IgA, or IgA2. Typical IgG molecule consists of two heavy chains γ and two identical light chains (λ or κ). Disulfide bonds link the light chain and the heavy chain therebetween, and link the heavy chains each other. The constant domain of the light chain pairs with the first constant domain of the heavy chain, the variable domain of the light chain (VL) pairs with the variable domain of the heavy chain (VH), thereby forms an antigen recognition site (agretope).
Variability of the variable domains (VL or VH) in the whole domain is not evenly distributed, and is generally concentrated in three segments called hypervariable regions. The more conservative parts in the variable domains are called the framework regions (FR). Each variable domain of native heavy and light chains comprise four FRs, the FR mainly form β-sheet structures connected by three hypervariable regions, which form the connecting β-sheet structure, and in some cases form part of a ring of β-sheet structure. The hypervariable regions in each chain is close to each other through the FR, and form an antigen binding site of an antibody together with hypervariable regions in another chain. See Kabat et al., SEQUENCES OF PROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991).
Epidermal growth factor receptor (EGFR, HER1, c-ErbB1) is a transmembrane glycoprotein composed of 1186 amino acid residues, with a molecular weight of 170 kD. EGFR has three parts: extracellular region, transmembrane region, and intracellular region (Jorissen R N, Walker F, Pouliot N, et al., Epidermal growth factor receptor: mechanisms of activation and signaling. Exp Cell Res, 2003; 284: 31-53). EGFR belongs to tyrosine kinase receptor subfamily type I (ErbB 1-4), having a tyrosine kinase activity. EGFR is stably expressed in many epithelial tissues, and mesenchymal and neurogenic tissues. EGFR are also highly expressed in solid tumors occurred in different organs, such as head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer, etc. (Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol, 2003; 21: 2787-2799). Growth factors such as transforming growth factor α (TGFα) and epidermal growth factor (EGF) are ligands for EGFR. The combination of these ligands with EGFR results in dimerization of EGFR, which activates an intracellular protein tyrosine kinase activity of the receptor, the C-terminal phosphorylation of specific tyrosine residues to provide binding sites for the intracellular signal transduction factor, thereby initiating multiple signal transduction pathways such as Shc, Grb2, Ras/MAPK, PI 3K and JAKs/STATs (Mendelsohn J, Baselga J. Status of epidermal growth factor receptor antagonists in the biology and treatment of cancer. J Clin Oncol, 2003; 21: 2787-2799; and Olayioye M A, Neve R M, Lane H A, et al. The EerbB Signaling network: receptor heterodimerzat ion in development and cancer. The EMBO J, 2000; 19: 3159-3167). EGFR regulates growth and differentiation of normal cells, increases invasiveness of tumor cells, promotes angiogenesis, inhibits apoptosis of tumor cells through mediating these pathways. (Castillo L, Etienne-Grimaldi M C, Fischel J L, et al. Pharmacological background of EGFR targeting Ann Oncol, 2004; 15: 1007-1012). For the characteristics of EGFR such as highly expression in tumors and important roles in tumor cell growth and differentiation, EGFR become promising targets for cancer diagnosis and treatment.
In recent years, there is growing evidence proving that the epidermal growth factor receptor (EGFR) is relevant with the occurrence and development of many tumors. In a variety of solid tumors, EGFR expression rate in head and neck cancer is the highest up to 95%-100%. Colorectal cancer is the second, with the expression rate of 72%-89%. EGFR-positive tumors have features of high malignancy and strong invasion, and EGFR expression levels correlated with prognosis. Thus it also becomes an important target for the current molecularly targeted cancer therapy. There is some evidence to verify that HER1/EGFR is expressed abnormally in solid tumors, and its clinical manifestations is transfer, shortened survival, poor prognosis, and well tolerations to chemotherapy and hormone therapy. After blocking HER1/EGFR, tumor formation can be inhibited, while the above-mentioned condition can be improved.
EGFR targeted drugs currently used to treat cancer can be divided into two categories: EGFR monoclonal antibodies and small molecule tyrosine kinase antagonist compound. Tyrosine kinase antagonist is mainly a small molecule quinolone-based compounds, which can competitive inhibit binding of ATP and the intracellular tyrosine kinase domain of EGFR, thereby affect the phosphorylation of tyrosine residues and inhibit EGFR downstream signal transduction.
EGFR monoclonal antibody competitively binds EGFR with the endogenous ligand, and produces anti-tumor effects by inhibiting activation of tyrosine kinase and promoting internalization of the EGFR. Presently, there are three kinds of anti-EGFR monoclonal antibody available for market at home and abroad. Compared with other chemotherapy drugs, these antibodies have higher specificity, lower side effects and achieved good results in clinical therapy. EGFR is a mature target for antibody drug development. As one of the three most popular mature target for an anti-tumor antibody drug development (HER2, EGFR, VEGF), the status of targeted therapy medicaments relevant to EGFR target including antibodies medicaments in cancer therapy is very important. Because of the limitations of currently commercially available anti-EGFR antibody medicaments, research and development of novel anti-EGFR antibody medicaments with high efficiency and low toxicity has been the focus of international pharmaceutical industry research.
In vitro assays and in vivo animal tests showed, on one hand, EGFR monoclonal antibody Erbitux (cetuximab) binds with EGFR to block phosphorylation, inhibit ligand-activated EGFR tyrosine kinase activity, and promote endocytosis and degradation of EGFR, thereby inhibit tumor cell growth and induce apoptosis thereof; on the other hand, Erbitux (cetuximab) inhibit tumor cell growth by recruiting cells to the tumor surface, such as natural killer (NK) cells, etc., and further by cytotoxicity (Bleeker W K, Lammerts van Bueren J J, van Ojik H H, Gerritsen A F, Pluyter M, Houtkamp M, Halk E, Goldstein J, Schuurman J, van Dijk M A, van de Winkel J G; Parren P W. Dual mode of action of a human anti-epidermal growth factor receptor monoclonal antibody for cancer therapy. J Immunol 2004; 173: 4699-707).
In addition, it can be found from the summary of research results on antibody medicaments molecules and mechanism thereof during the therapy of diseases that, the therapeutic role of various anti-EGFR monoclonal antibodies in the body is not only relative with their affinity to EGFR, but also with the ADCC activity. (Patel D, Guo X, Ng S, Melchior M, Balderes P, Burtrum D, Persaud K, Luna X, Ludwig D L, Kang X. IgG isotype, glycosylation, and EGFR expression determine the induction of antibody-dependent cellular cytotoxicity in vitro by cetuximab. Hum Antibodies. 2010; 19: 89-99).
Furthermore, on the basis of without changing the three-dimensional structure of an antibody, antibody engineering technology can achieve conversion among IgG antibody subtypes (Z Steplewski, L K Sun, C W Shearman, J Ghrayeb, P Daddona, and H Koprowski. Biological Activity of Human-Mouse IgG1, IgG2, IgG3, and IgG4 chimeric monoclonal Antibodies with antitumor specificity. PNAS 1988; 85: 4852-4856).
Currently, with the development of technology, therapeutic monoclonal antibody is divided into three species: chimeric antibody sequences of about 70% human origin, humanized antibodies having 90-94% humanized sequences, as well as whole humanized antibody having 100% human sequence. The ratio of human sequence suggests the possibility of potential immunogenicity caused when the antibody is used for human treatment, therefore, the possibility of fully human antibody producing immunogenicity is lower than that of chimeric antibodies, and fully human antibody is better than chimeric antibody and humanized antibody when use as therapeutic medicaments.
ADCC (antibody-dependent cell-mediated cytotoxicity) refers to NK cells, macrophages and neutrophils which express IgG-Fc receptor, kill these target cells, by binding to Fc fragments of IgG antibody which have already been bound on surfaces of target cells such as virus-infected cells and tumor cells. IgG antibody can mediate ADCC function of these cells, wherein NK cells are major cells to show ADCC. In the occurrence of antibody-mediated ADCC action, the antibody can only specifically bind to the corresponding epitope on the target cells, effector cells such as NK cells can kill any target cells bound with the antibodies, so the binding of antibodies to antigens on the target cells is specific, while cytotoxicity to target cells by NK cells and other cells is non-specific.
Several anti-EGFR monoclonal antibody drugs already on the market still have many deficiencies. For example, Erbitux is a human chimeric IgG1 antibody, which has a considerable degree of immunogenicity, prone to human anti-mouse antibody response, worse side effects, thus affecting the efficacy thereof. Panitumumabhas a fully human IgG2 subtype, but it lacks of ADCC-relative biological activity, relies solely on blocking the EGFR signaling pathway to inhibit tumor growth, without another ADCC mechanism on inhibition of tumor, and thus the anti-tumor effect thereof is weak.
Accordingly, there remains a need for improved anti-EGFR antibodies to inhibit tumor. The present inventors have surprisingly found that by use of antibody engineering technology platform, panitumumab is changed from IgG2 subtype to IgG1 subtype, a fully human anti-EGFR antibody molecule with new sequence is formed, and the antibody molecule has the same target-binding characteristics with panitumumab, and because of different antibodies subtypes, the antibody molecule has a stronger ADCC biological activity, and a stronger anti-tumor effect compared with panitumumab. In addition, since the antibody is a fully human antibody molecule, which has a lower immunogenicity than that of the chimeric antibody Erbitux, it has lower potential clinical side effects. Thus, antibodies of the present disclosure brings together the advantages of antibodies known in the prior art and obtain unexpected technical effect.